Developing adapted wheat lines with broad-spectrum resistance to stem rust: Introgression of Sr59 through backcrossing and selections based on genotyping-by-sequencing data

Control of stem rust, caused by Puccinia graminis f.sp. tritici, a highly destructive fungal disease of wheat, faces continuous challenges from emergence of new virulent races across wheat-growing continents. Using combinations of broad-spectrum resistance genes could impart durable stem rust resistance. This study attempted transfer of Sr59 resistance gene from line TA5094 (developed through CSph1bM-induced T2DS·2RL Robertsonian translocation conferring broad-spectrum resistance). Poor agronomic performance of line TA5094 necessitates Sr59 transfer to adapted genetic backgrounds and utility evaluations for wheat improvement. Based on combined stem rust seedling and molecular analyses, 2070 BC1F1 and 1230 BC2F1 plants were derived from backcrossing BAJ#1, KACHU#1, and REEDLING#1 with TA5094. Genotyping-by-sequencing (GBS) results revealed the physical positions of 15,116 SNPs on chromosome 2R. The adapted genotypes used for backcrossing were found not to possess broad-spectrum resistance to selected stem rust races, whereas Sr59-containing line TA5094 showed resistance to all races tested. Stem rust seedling assays combined with kompetitive allele-specific PCR (KASP) marker analysis successfully selected and generated the BC2F2 population, which contained the Sr59 gene, as confirmed by GBS. Early-generation data from backcrossing suggested deviations from the 3:1 segregation, suggesting that multiple genes may contribute to Sr59 resistance reactions. Using GBS marker data (40,584 SNPs in wheat chromosomes) to transfer the recurrent parent background to later-generation populations resulted in average genome recovery of 71.2% in BAJ#1*2/TA5094, 69.8% in KACHU#1*2/TA5094, and 70.5% in REEDLING#1*2/TA5094 populations. GBS data verified stable Sr59 introgression in BC2F2 populations, as evidenced by presence of the Ph1 locus and absence of the 50,936,209 bp deletion in CSph1bM. Combining phenotypic selections, stem rust seedling assays, KASP markers, and GBS data substantially accelerated transfer of broad-spectrum resistance into adapted genotypes. Thus, this study demonstrated that the Sr59 resistance gene can be introduced into elite genetic backgrounds to mitigate stem rust-related yield losses.


Introduction
Wheat (Triticum aestivum L.) is an important source of calories and protein in the daily human diet world-wide [1].Due to the current rapid growth in the global population, a 60% increase in wheat production will be necessary in order to maintain its current share of the human diet by 2050 [2].Wheat yield will need to be increased by at least 2% each year to meet this demand, a target that is currently not being attained [3].The major constraints to achieving the necessary yield increase are biotic and abiotic stresses that impair crop performance, with rust diseases in particular having the potential to cause yield losses in severe outbreaks.Among these diseases, stem rust (caused by the fungus Puccinia graminis f. sp.tritici (Pgt)) is a major threat to wheat production across many regions of the world, because it is capable of causing severe yield loss [4].Although fungicide application can effectively manage stem rust, it is associated with drawbacks such as high costs, significant environmental impact, and negative effects on human health [5].Hence, genetic resistance is the most economical and environmentally sustainable control measure to protect wheat yields from the threat of stem rust.The frequent emergence of new Pgt races is a major challenge to success in breeding resistance to this pathogen in wheat.An example of this is emergence of the Ug99 race group, which is capable of overcoming all known and extensively deployed stem rust (Sr) resistance genes, including Sr24, Sr31, Sr36, and SrTmp.This constant adaptation of the pathogen has increased concerns about global epidemics [6,7].
Other widely virulent Pgt races, such as TRTTF, TKTTF, TTRTF, TTKST, PRCTM, and TTKTT, have been found to possess additional virulence combinations, including virulence to Sr22+Sr24, Sr24+Sr31, Sr13b+Sr35+Sr37, and Sr24+Sr31+SrTmp genes [8][9][10].The emergence of these novel races and their spread into Europe is alarming, since stem rust disease has largely been absent for nearly 60 years [11][12][13].Moreover, a high proportion of cultivars grown in Europe are susceptible to these emerging races, e.g., ~80% of wheat cultivars currently grown in the United Kingdom are susceptible to race TKTTF [11], while resistance genes such as Sr24, Sr31, and Sr38, present in German wheat cultivars are limited in their effectiveness against these novel races of Pgt [14].Of the currently known and described wheat Sr genes, 35 out of 73 derive from the primary gene pool of wheat and the majority of these do not confer broad-spectrum resistance [4].Until recently, the Sr31 resistance gene was considered highly effective in conferring broad-spectrum resistance against all Pgt races, and was introduced into many wheat cultivars over the past 30 years [7].
A large number of wheat-rye introgression lines were developed in the 1980s-2000s by the late Professor Arnulf Merker at the Swedish University of Agricultural Sciences [31,32].Some of these lines were used in field and greenhouse screenings to identify the line 'SLU238' [2R (2D) wheat-rye disomic substitution], which was found to confer broad-spectrum resistance to all Pgt races tested [33].TA5094, a line derived from 'SLU238', has since been shown to possess a T2DS�2RL Robertsonian translocation with a stem rust resistance gene designated Sr59 [16].Due to the lack of acceptable agronomic performance in TA5094 based on the CSph1bM background, there is an urgent need to transfer this gene to a more suitable genetic background and evaluate its potential use in wheat resistance breeding.This paper describes transfer and subsequent evaluation of Sr59 to agronomically suitable genetic background derived lines through: 1) marker-assisted backcross breeding; 2) stem rust seedling assessment; 3) background selection; and 4) physical mapping of the Sr59 resistance gene on chromosome 2RL.

Population development, stem rust seedling evaluations, and molecular marker analysis
The F 1 plants obtained from crosses between line TA5094 and the recurrent parents (BAJ#1, KACHU#1, and REEDLING#1) were backcrossed to each of the corresponding recurrent parents, generating BC 1 F 1 seeds.A total of 2,070 BC 1 F 1 plants were assessed for their seedling responses to Pgt race TTTTF (isolate 01MN84A-1-2), in trials at the USDA-ARS Cereal Disease Laboratory and University of Minnesota using a previously described stem rust seedling assay [34,35].For each recurrent parent, 94 resistant BC 1 F 1 plants were selected (i.e., in total 282 plants) and analyzed for the presence of Sr59 by use of three KASP markers: KASP_2RL_c25837C1, KASP_2RL_c21825C1, and KASP_2RL_c20194C2 [16].Based on the results of KASP marker analysis, BC 1 F 1 plants with Sr59 were selected and used for backcrossing to produce BC 2 F 1 plants.The backcross generated 1,230 BC 2 F 1 plants, which were assessed against Pgt race TTTTF.Resistant plants were selected and tested for the presence of Sr59 with the three KASP markers.Plants carrying Sr59 were selfed to produce the BC 2 F 2 generation, which resulted in a total of 846 families (from all recurrent parents).These families were again evaluated against Pgt race TTTTF and resistant plants were selected and checked with the three KASP markers.The BC 2 F 2 generation was also evaluated using race TTKSK, and the pattern of segregation was analyzed.From the BC 2 F 2 , additional generations (BC 2 F 3 , BC 2 F 4 , and BC 2 F 5 ) were created through selfing, 20 plants from each generation were selected based on the seedling response to race TTTTF, and presence of Sr59 was validated by KASP markers.In addition, 10-15 BC 2 F 4 and BC 2 F 5 plants from each family were tested for their seedling response to races TTKSK, TTTSK, and TRTTF.The BC 2 F 5 families were also assessed against races TPMKC, QTHJC, RKQQC, and RCRSC.The segregation pattern data were assessed using chi-square (χ 2 ) analysis.

Genotyping and data analysis
The population was genotyped using GBS as described previously [36].Tissue sampling and DNA extraction were carried as described previously [37].Approximately 10 cm of young leaf tissue from each of the donor parents (TA5094 and CSph1bM), recurrent parents, and a total of 128 selected BC 2 F 2 plants (from all three recurrent parents) were collected in a 96-well tissue collection plate.Genomic DNA was isolated using the Qiagen BioSprint 96 instrument and the associated Qiagen BioSprint DNA Plant kit (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/ biosprint-96-dna-plant-kit/#orderinginformation).DNA sequencing libraries were prepared and sequenced at the University of Minnesota Genomics Center.In brief, the isolated DNA was quantified with PicoGreen for GBS genotyping and normalized to 20 ng/μL.The GBS libraries were prepared in 96-plex using two restriction enzymes: a rare cutter PstI (5 0 -CTGCAG-3 0 ) and a frequent cutter MspI (5'-CCGG-3') with a common reverse adapter ligated [36,38].Libraries were sequenced on Illumina HiSeq2500 (Illumina, San Diego, CA, USA).Sequences obtained in the FASTQ files were passed through a quality filter of Q >30 and then de-multiplexed to obtain reads for each individual.Thereafter, the GBS reads were aligned to the International Wheat Genome Sequencing Consortium (IWGSC) Reference Sequence v1.0 (RefSeq v1.0) assembly and Rye Genome Sequencing Consortium Reference Sequence, using the Burrow-Wheelers Alignment tool (BWA) v0.7.4 [39].Marker discovery, i.e., identification of SNPs, was accomplished using Samtools+Bcftools [40].SNPs with minor allele frequency (MAF) <5% and more than 20% missing data were removed.After processing, 40,584 SNP markers for wheat and 15,116 SNP markers for chromosome 2R were retained for further analyses.Allele frequencies and genetic relationship between donor and recurrent parental lines were calculated using TASSEL v5.2.65 [41].Principal component analysis (PCA) was performed using the function 'prcomp' in R 4.0.2.MapChart 2.2 (https://www.wur.nl/en/show/mapchart.htm) was used to draw physical maps.

Selection of plants for recovery of recurrent parents
Progeny lines were selected based on their phenotypic and genomic similarity to the recurrent parents and used in the next backcross generation.The phenotypic parameters of each backcross generation (BC 1 F 1 and BC 2 F 1 ) were evaluated in the greenhouse, to determine whether the plants were similar to the recurring parents based on their height, tillering, heading date, flowering, spike characteristics (with or without awns), seed fertility, and maturity day.Background selection for alleles similar to those of the recurrent parents was then performed on the BC 2 F 2 generation, using GBS markers distributed across all 42 wheat and the 2R rye chromosomes.Next, individual plants from each generation (BC 2 F 3 to BC 2 F 6 ) were carefully selected based on highest phenotypic and genotypic similarities to the recurrent parents, ensuring consistent inheritance of desired traits across generations.
BC 2 F 7 and BC 2 F 8 lines resistant to races TTTTF and TTKSK were sown on the field of Lantma ¨nnen Research Station in Svalo ¨v (55.925621˚N, 13.096742˚E) for phenotyping evaluations.In one replicate field evaluation, these lines were sown in small plots to assess phenotypic traits compared with the recurrent parents.Data were collected on characteristics such as number of days to 50% flowering and maturity, plant height, tillering, lodging, susceptibility to diseases (e.g., rusts, powdery mildew, septoria, FHB, etc.), and grain color.

Stem rust seedling evaluations and marker-assisted backcrossing
Evaluation of the BC 1 F 1 and BC 2 F 1 populations using race TTTTF suggested presence of a major resistance gene following crossbreeding with BAJ#1 (Table 2).The P-values for all other BC 1 F 1 and BC 2 F 1 populations resulting from these crosses were <0.05, indicating a segregation pattern deviating from the expected 1:1 ratio.Such deviation suggests potential segregation distortion or the involvement of more than one major gene in resistance.However, the segregation ratio was close to 3:1 (P>0.05) for the BC 2 F 2 populations from the backcrosses to BAJ#1 and REEDLING#1 (Table 2), indicating a single dominant major resistance gene.To verify presence of Sr59 in plants that were selected for further generations, a number of resistant plants from each of the BC 1 F 1 , BC 2 F 1 , and BC 2 F 2 populations were selected and genotyped with three KASP markers (Table 2).Plants found to contain the gene were transplanted for backcrossing and selfing (Table 2).The BC 2 F 2 populations were also phenotyped with race TTKSK, which resulted in a significant deviation from the expected 3:1 segregation ratio (P<0.01).Instead, the segregation ratio was closer to 14:2 (P>0.05),suggesting presence of a major gene plus one or more additional genes co-acting with the major gene.The BC 2 F 4 and BC 2 F 5 populations, obtained through assessments against race TTTTF and subjecting selected resistant plants to KASP genotyping, showed a similar high level of resistance (IT; 1-to; 11+) against races TTTSK, TTKST, and TRTTF as seen in their resistant parental lines (Table 1), indicating successful transfer of Sr59 to the later generations.The BC 2 F 5 families also displayed similar responses (IT; 01-to; 11+) as SLU238 and TA5094 when evaluated against races TPMKC, QTHJC, RKQQC, and RCRSC.

GBS genotyping and physical location of Sr59 on the 2R chromosome
Through alignment of raw GBS reads against the rye line 'Lo7' (International Rye Genome Sequencing Consortium (IRGSC)) reference genome), followed by filtering to remove SNPs with missing values �20% and minor allele frequency (MAF) � 5%, the physical positions of the 15,116 SNPs obtained were mapped to chromosome 2R at 347,694 bp to 945,773,747 bp (  tested in the BC 2 F 3 lines, where the homozygous lines showed IT; 1 (100% red color), while the segregating lines showed; 1 to 3+4 (varying percentages of green and red color) (Fig 2C)

Recovery of the recurrent parent (background selection)
To select lines that resembled their recurrent parents as much as possible (with the exception of the addition of Sr59), a total of 40,584 SNPs across all 21 chromosomes were used to identify the most suitable lines in the BC 2 F 2 population.Based on polymorphic SNPs, whole-wheat genome PCA identified five distinct clusters: 1) CSph1bM and CSA; 2) SLU238 and TA5094; 3) BC 2 F 2 BAJ#1 population; 4) BC 2 F 2 KACHU#1 population; and 5) BC 2 F 2 REEDLING#1 population (Fig 4).The PCA results showed that most of the BC 2 F 2 of a recurrent parent clustered at the same plot, indicating genomic recovery of the recurrent parent genome.As shown in Table 3, percentage genome recovery for the recurrent parents ranged from 66% to 75% across the three BC 2 F 2 populations.The plants with the highest recurrent parent genome recovery and carrying Sr59 were selected to generate homozygous lines through selfing.

Phenotypic selection in greenhouse and field conditions
Besides using SNPs to produce lines resembling recurrent parental lines, selection was carried out in greenhouse and field evaluations with plant height and awns/awnless spikes being key phenotypic characters considered during crossing and backcrossing, as these characters differed between SLU238 and TA5094 compared with the recurrent parents, BAJ#1, KACHU#1, and REEDLING#1 (Table 2).Thus, 40 BC 1 F 1 plants with maximum phenotypic similarity to the recurrent parents were used for developing the BC 2 F 1 population.In BC 2 F 2 to BC 2 F 6 populations, plants were selected based on four characteristics: plant height, awns/awnless, days to maturity, and seed fertility.BC 2 F 7 lines showing a homozygous resistance reaction to Pgt race TTTTF and positive KASP marker data were sown in the field in 2020.The following phenotypic parameters were considered when selecting single plants in the field in 2020: plant stand, tillering, plant height, awns/awnless spikes, lodging, days to maturity, and seed fertility.Following the phenotypic analysis described above, BC 2 F 8 populations were sown in the greenhouse and selfed to produce another generation, and BC 2 F 9 populations were planted in the field in spring 2021 to select plants whose phenotypic similarity to the recurrent parents was greatest.The three KASP markers (c20194_115, c25837_157, and c21825_230) were used again to verify presence of the Sr59 resistance gene in the individual BC 2 F 7 to BC 2 F 9 plants.

Ph1 allele status
A BLAST search against the IWGSC reference sequence v1.0 resulted in 2,050 GBS SNPs annotated in the range 16,637-712,890,017 bp on chromosome 5B (Fig 5).Furthermore, a deletion breakpoint of 50,936,209 bp (51 Mb), located from 396,630,846 bp to 447,567,055 bp, was detected in line CSph1bM, indicating the position of the Ph1 locus on the 5B chromosome (Fig 5).The GBS data also revealed presence of the Ph1b deletion in TA5094 and two BC 2 F 2 populations (31-Kachu and 34-Kachu), whereas it was not detected in the other BC 2 F 2 populations, SLU238, CSA, or the recurrent parents.These results show that most of the BC 2 F 2 populations carry the Ph1 allele, and that the status of 2DS.2RL (and thus the introgression of Sr59 into the wheat genetic background of the recurrent parents) is stable.

Discussion
In this study, the Sr59 stem rust resistance gene was transferred, using TA5094 as a donor of rye chromatin, into three elite wheat lines, through marker-assisted backcrossing selection and stem rust seedling screening (BC 1 F 1 to BC 2 F 5 ).Seedling screening and KASP marker analysis allowed us to trace presence/absence of Sr59 from the parental lines in all progeny through all generations.The use of high-throughput genomic tools, such as GBS, facilitated the application of a strong selection pressure that increased the probability of recovering the elite recurrent parent background genome, while at the same time preserving the translocation fragment 2DS.2RL containing Sr59.The GBS background selection accurately identified both the translocated 2DS.2RL and the deletion region on 5BL (ph1b deletion).Sr59 contributed stable resistance, as demonstrated by seedling screening against multiple Pgt races and KASP marker analysis across all populations developed.Thus our novel GBS-and marker-assisted method was able to eliminate the ph1b deletion while transferring Sr59 into the genetic background of three widely adapted wheat cultivars (BAJ#1, KACHU#1, and REEDLING#1) from CIMMYT through backcrossing, and can ultimately produce wheat lines suitable for breeders.Initially, the Sr59 resistance gene was selected  based on the stem rust seedling response and marker-assisted backcrossing selection.Previous studies have shown that two backcrossing generations can recover approximately 87.5% of the recurrent parent genome [41].Use of a large backcrossing population is common practice to introgress resistance genes from an alien genome into an elite background [25].Aside from marker-assisted backcrossing, we used GBS genotyping to select plants with the greatest amount of the recurrent parent genome.Average recurrent parent genome recovery of 71.2%, 69.8%, and 70.5% was observed in the BC 2 F 2 populations (Table 3).This fairly low recovery might be because of a less-than-optimal representation of 2DL in the GBS dataset, due to the fact that we started off with a 2DS.2RL translocation in the donor parent (Table 3).In GBS genotyping, the A and B genomes are reported to have the highest number of SNPs, while the D genome has the lowest number [42].Evolutionary history and gene flow may be the reason for the poor D genome representation [43].In previous studies, GBS has been found to be an inexpensive and robust approach for genotyping crop genomes, as it enables discovery of a high number of genome-wide markers, often SNPs [38].Several studies have demonstrated that GBS detects small introgressions in wheat and barley [44,45].In the present, we study aligned GBS reads and located the physical positions of SNPs in wheat and 2R rye chromosomes based on the reference genome RefSeq v1.0 and the International Rye Genome Sequencing Consortium.In this alignment, GBS demonstrated the physical positions of 40,584 SNPs across the wheat genome and 15,116 SNPs for the 2R chromosome.Through this highthroughput genotyping procedure, it was possible to detect both translocation lines and nontranslocation lines in the BC 2 F 2 populations.We also used PCA to visualize the grouping of lines based on their genetic relationship, i.e., based on the differences in 2RL segments transferred to the BC 2 F 2 progeny (Fig 3).Basically, the susceptible BC 2 F 2 plants and parental lines without chromosome 2R (i.e., CSph1bM, BAJ#1, KACHU#1, REEDLING#1) clustered with a positive PCA1 (Fig 3).Likewise, 24 BC 2 F 2 resistant plants grouped closely to the lines TA5094 and SLU238, indicating presence of the whole 2RL chromosome segment (Fig 3).Additionally, a total of 28 BC 2 F 2 resistant plants clustered differently, indicating that these lines most likely resemble each other as regards their genomic composition, for both wheat and rye genome segments (Fig 3).Some factors, such as chromosomal segment rearrangements, segmental duplications, and differences in recombination frequencies caused by genomic structural variations, may explain the marker orders observed in this short segment.The introgression of rye chromosomes into wheat genomes can result in structural changes and rearrangements, as the heterochromatin DNA of rye chromosomes can interfere with chromosome synapsis [2,46].There may have been chromosome rearrangements in the BC 2 F 2 population that resulted in a shorter 2RL segment than in the other 24 BC 2 F 2 lines.This study showed that the Sr59 resistance gene in the 2RL chromosome segment is stable for normal transmission through the male gamete, preventing segregation distortion in cultivar development.No segregation distortion was observed in any of the three populations evaluated, either for race TTTTF or for race TTKSK.However, previous studies have shown that segregation distortion is a common feature when alien chromosomes are introgressed in the wheat genome [25].Development of chromosome-specific SNP markers covering target chromosomes and facilitating homologous recombination on chromosomes containing resistance genes can assist in tracking rye resistance genes within wheat more effectively by minimizing chromosome transfer and reducing the likelihood of linkage to undesirable alleles.Line SLU238 wheat-rye disomic substitution carrying 2R (2D) chromosome exhibits effective resistance to several virulent races of stem rust and has been used to develop 2DS.2RL wheat-rye translocation lines [16].Substitution lines serve as bridging materials in the development of wheat-alien translocation lines [25].By incorporating distinct alien chromosome segments with desired traits through chromosome translocations, linkage drag can be reduced [25,28].
For the three KASP markers (c20194_115, c25837_157, and c21825_230), BLASTN was used and their positions were mapped at 914,812,229 bp to 943,109,279 (28 Mb), as physically mapped in a previous study [16].Based on the GBS reads, we observed a deletion spanning from 396,630,846 Mb to 447,567,055 Mb on chromosome 5B in TA5094 and two BC 2 F 2 plants (31-Kachu and 34-Kachu), indicating deletion of the CSph1bM allele.A deletion on the 5B chromosome was not detected in the other BC 2 F 2 plants, all carrying the Ph1 allele, preventing true homology in pairing.New ph1b deletion-specific markers based on 90K SNPs have been developed to accurately identify the ph1b deletion region [47].Successful cross-over between the wheat chromosome and its wild relatives is challenging, which is why CSph1bM mutants are recommended for inducing meiotic homoeologous recombination [23].Several resistance genes, such as Sr32, Sr47, Sr39, Sr59, and Yr83 have been successfully transferred using CSph1bM mutants [16,24,25,29].This approach provides an effective means of introducing beneficial traits from wild relatives for wheat improvement.The Sr11 and Sr38 resistance genes were postulated to be present in three recurrent parents from CIMMYT (Table 1).The Sr11 and Sr38 resistance genes have previously been reported to be widely prevalent in wheat cultivars worldwide [48,49], and the recurrent parents may carry both genes.Due to the limited agronomic performance of the CSph1bM mutant and the elimination of the ph1b allele in line TA5094, it is necessary to transfer Sr59 into adapted cultivars.Here, we successfully transferred Sr59 from TA5094 to the genetic background of the three cultivars BAJ#1, KACHU#1, and REEDLING#1.The progeny showed proven resistance to TTTTF (USA), TKTTF (Ethiopia), QTHJC, TPMKC, RKQQC, RCRSC, TTKST, TTKSK, TTTSK, TRTTF, and LTBDC (Australian Pgt race 98-1,2,3,5,6).Following backcrossing, morphological and agronomic characteristics with the greatest similarity to the recurrent parents were considered.Other resistance genes such as SrTA1662, Yr15, and Sr39 have been transferred in previous work using backcrossing to recover the recurrent parent phenotype [25,50,51].In conclusion, Sr59 offers broad-spectrum resistance to stem rust races, making it a valuable gene for wheat improvement, while the reliability of KASP markers for Sr59 makes them suitable for markerassisted selection of stem rust resistance in wheat breeding.These findings can facilitate further production of stem rust-resistant wheat cultivars, developed with Sr59 resistance in their elite background, which can act as additional assets for improving wheat yields and preventing stem rust losses.

Fig 1 )
. In Fig 2B, the physical positions of the 15,116 SNPs from GBS (rye alleles) are shown in red color, whereas blue color indicates the presence of wheat alleles when mapping the BC 2 F 2 populations.The physical positions of three KASP markers (c20194_115, c25837_157, and c21825_230) and the GBS results showed that Sr59 was located in the 2RL segment (Fig 1).The three KASP markers were also used to track the Sr59 introgression into the recurrent parent's background through the crossing scheme utilized in the present study.BLASTN searches of the three KASP markers against IRGSC positioned the Sr59 resistance gene between 914,812,226 bp and 943,109,279 bp on chromosome 2RL (Fig 1).Presence of 2RL was also clearly verified in lines SLU238, SLU239, and TA5094, as demonstrated by the red color in Fig 2B, whereas 2D (wheat) was verified in BAJ#1, KACHU#1, REEDLING#1, CSA, CSph1bM, and susceptible lines (6-BAJ-S, 23-KACHU-S, 33-Reed-S, 35-Reed-S, and 45-Reed-S), as demonstrated by the blue color in Fig 2B.A strong association was observed between presence of the rye/wheat alleles determined by the GBS dataset and resistance (red color)/susceptibility (green color) reactions to race TTTTF and TTKSK (Fig 2A and 2C).Most of the BC 2 F 3 lines showed homozygosity (100% red color) for resistance to the TTTTF race, although some lines segregated (40% red and 60% green; Fig 2A).The reaction to race TTKSK was

. The BC 2 F
3 lines are shown as 100% green color for both races (TTTTF and TTKSK) in Fig 2A and 2C.PCA analysis based on the results of the GBS data (15,116 SNPs on chromosome 2R) clustered the genotypes evaluated into five distinct clusters: A) SLU238, SLU239, and TA5094; B) The BC 2 F 2 population consists of recurrent parents harboring the 2RL chromosome proximate to SLU238 and TA5094; C) BC 2 F 2 population derived from recurrent parents carrying the 2RL chromosome; D) Recurrent parents (BAJ #1, KACHU #1, and REEDLING #1) and the susceptible BC 2 F 2 population without 2RL; and E) CSph1bM and CSA (Fig 3).

Fig 1 .
Fig 1. Location of the Sr59 resistance gene on rye chromosome 2R, determined using GBS reads aligned to the rye line 'Lo7'.Dark red denotes presence of physical SNPs, while white denotes absence of SNPs throughout the 2R chromosome.The trio of SNPs on 2RL correspond to the three KASP markers identified previously.https://doi.org/10.1371/journal.pone.0292724.g001

Fig 2 .
Fig 2. Physical location of rye chromosome 2R based on GBS data and seedling responses to stem rust races TTTTF and TTKSK.a) Seedling assay for race TTTTF in the BC 2 F 3 population, where red denotes resistance and green susceptibility; b) physical positions of the 15,116 SNPs from GBS reads in the BC 2 F 2 population, where red denotes the rye allele and dark blue the wheat allele; c) seedling assay for race TTKSK in the BC 2 F 3 population, where red denotes resistance and green susceptibility.https://doi.org/10.1371/journal.pone.0292724.g002